DNA condensation is in all living organisms a difficult process due to the large size of a genome, in which as much genetic information as possible has to be packaged into a small volume. Frequently, the storing compartment has a diameter, which is thousand times smaller than the length of the DNA molecule. Evolution has solved this problem in different ways. Overlapping genes in viruses are able to increase the genomic information without compromising the size of the DNA molecule. In other viruses positively charged molecules and cations guarantee a neutralization of the DNA polymer and hence dense packaging. In higher organisms proteins are employed in tightly packaging DNA aggregates. An interesting example for successful condensation and packaging of a long dsDNA genome in a small volume is presented by the Chlorella viruses. In the case of virus PBCV-1 (Paramecium bursaria Chlorella virus-1), the prototype of Chlorella viruses, a ca. 100 µm long DNA molecule is packaged into a volume of only 1.7 * 10-7 m3. The small amounts of polyamines, which are present in the viral particle, are insufficient for charge compensation. To test whether virus PBCV-1 uses, like phages, cations for charge compensation, we measured the content of cations in the virus particles with Energy dispersive X-ray spectroscopy and Inductively coupled plasma-mass spectrometry. The data reveal that cations can be detected, but the concentration in the particle is not sufficient to accomplish the whole neutralization of the DNA molecule; only one fifth of the total phosphor concentration can be neutralized by Mg2+ or K+ or Ca2+. By summing up all cations, detected in the particle, we can estimate, that 58 % of the total phosphate can be neutralized in this way.
Imaging of ejected viral DNA indicates that it is intimately associated with proteins in a periodic fashion. A combination of fluorescence images of ejected DNA and a bioinformatics analysis of the DNA reveal periodic patterns in the viral DNA. The periodic distribution of GC rich regions in the genome provides potential binding sites for basic proteins. Collectively the data indicate that the large Chlorella viruses have a DNA packaging strategy that differs from that of bacteriophages; it involves proteins and shares similarities to that of chromatin structure in eukaryotes.
In the genome of PBCV-1 an ORF, A278L, encodes for a protein, with kinase activity, which resembles in its physical properties, i.e. small size and basic isoelectric point (IP: 10.8), histones. Further, the protein is present with a high copy number (397) in the virus particle. Altogether, this makes the gene product of A278L an interesting candidate for DNA condensation. We find in a qualitatively way that the recombinantly produced and purified protein A278L indeed causes a higher degree of genomic condensation. In comparison with the neutral protein BSA, A278L causes a roughly two fold higher degree of DNA clustering. Atomic force microscopy force spectroscopy was used to determine the DNA binding capacity of the A278L protein. An analysis of binding of native viral proteins, which were in contact with the viral DNA after a release by osmotical shock from the PBCV-1 virions, revealed a characteristic binding behaviour. The data revealed an average force of 64 ± 23 nN for the binding between native proteins and viral DNA. The recombinant protein A278L showed a similar binding strength to isolated viral DNA; on average 22 ± 3 nN were required to separate the protein from the DNA. These measured forces for separating proteins from DNA are specific for a protein/DNA interaction because proteins could be pulled away from the mica surface with a 100 times smaller force. The results of these experiments imply that virus PBCV-1 employs small molecules such as cations and polymaines in combination with proteins for packaging its large dsDNA genome in the virus particle. The proteins may support the organization of meta-structures and the consequent achievement of a crystalline-like order inside the virion. In addition, they may also provide substantial contribution to the neutralization of the DNA. The present results provide some new understanding of viral DNA packaging. This may help to create artificial devices for DNA shuttles in the field of gene therapy.